Exposure of mammalian cells to ionizing radiation is known to induce chromosome instability (CIN), which can promote the rapid accumulation of genetic changes leading to cancer. We have demonstrated that DSBs occurring near the ends of chromosomes, called telomeres, can promote CIN by causing sister chromatid fusions that initiate breakage/fusion/bridge (B/F/B) cycles. B/F/B cycles occur when fused sister chromatids break during anaphase as their centromeres are pulled in opposite directions. Because breakage does not occur at the site effusion, one daughter cell acquires a chromosome with an inverted repeat at its end, while the other has a chromosome with a terminal deletion. This cycle is then repeated in subsequent cell cycles, resulting in further gene amplification and large terminal deletions. B/F/B cycles continue until the chromosome acquires a new telomere, which we have shown can occur by multiple mechanisms. One of the most common mechanisms is nonreciprocal translocation, which results in the transfer of instability to the chromosome donating the translocation due to the loss of its telomere. As a result, a single DSB near a telomere can result in the instability in multiple chromosomes. This proposal has two specific aims. In the first specific aim, we will address the hypothesis that sister chromatid fusions resulting from DBSs near telomeres result from a deficiency in nonhomologous end joining (NHEJ) near telomeres, as has been demonstrated in yeast. For these studies, we will create an assay system using isogenic cell lines to compare the efficiency of NHEJ at DSBs occurring at various distances from a telomere. We will also use this assay system to address differences in the repair proteins involved in NHEJ and sister chromatid fusion, which has been proposed to involve microhomology-mediated homologous recombinational repair. In the second specific aim, we will address the hypothesis that the p53 and Rb proteins involved in replicative senescence are important in preventing CIN due to DSB-induced telomere loss. In addition, we will establish a genetic screen using green fluorescent protein to monitor the duration of B/F/B cycles to identify additional proteins that can prevent B/F/B cycles. These studies are important in that they will provide new insights into mechanisms of chromosome instability in cancer and lead to new approaches for the development of therapies for preventing CIN in cancer cells.